Description and molecular characterisation of Babesia ailuropodae n. sp., a new piroplasmid species infecting giant pandas

Background Babesia spp. are protozoan parasites that infect the red blood cells of domesticated animals, wildlife and humans. A few cases of giant pandas (a flagship species in terms of wildlife conservation) infected with a putative novel Babesia sp. have been reported. However, comprehensive research on the morphological and molecular taxonomic classification of this novel Babesia sp. is still lacking. This study was designed to close this gap and formally describe this new Babesia sp. infecting giant pandas. Methods Detailed morphological, molecular and phylogenetic analyses were conducted to characterise this Babesia sp. and to assess its systematic relationships with other Babesia spp. Blood samples from giant pandas infected with Babesia were subjected to microscopic examination. The 18S ribosomal RNA (18S rRNA), cytochrome b (cytb) and mitochondrial genome (mitogenome) of the new Babesia sp. were amplified, sequenced and assembled using DNA purified from blood samples taken from infected giant pandas. Based on the newly generated 18S rRNA, cytb and mitogenome sequences, phylogenetic trees were constructed. Results Morphologically, the Babesia sp. from giant pandas exhibited various forms, including round to oval ring-shaped morphologies, resembling those found in other small canine Babesia spp. and displaying typical tetrads. Phylogenetic analyses with the 18S rRNA, cytb and mitogenome sequences revealed that the new Babesia sp. forms a monophyletic group, with a close phylogenetic relationship with the Babesia spp. that infect bears (Ursidae), raccoons (Procyonidae) and canids (Canidae). Notably, the mitogenome structure consisted of six ribosomal large subunit-coding genes (LSU1-6) and three protein-coding genes (cytb, cox3 and cox1) arranged linearly. Conclusions Based on coupled morphological and genetic analyses, we describe a novel species of the genus Babesia, namely, Babesia ailuropodae n. sp., which infects giant pandas. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s13071-024-06402-6.

caused by parasites are considered one of the significant factors affecting the health of giant pandas [3,4].
The genus Babesia comprises protozoan parasites that are transmitted by ticks; these parasites parasitise red blood cells in livestock, wild animals and humans [5,6].There are more than 120 Babesia species globally; these species can infect various hosts and induce babesiosis.Haemolysis, fever, jaundice, anaemia and haemoglobinuria are typical clinical signs of babesiosis [7].Babesiosis primarily affects mammals, including, but not limited to, members of the Canidae, Felidae, Ursidae, Procyonidae, Bovidae, Equidae and Cervidae families.The most common agents of babesiosis include Babesia canis, Babesia rossi, Babesia vogeli, Babesia conradae, Babesia gibsoni, Babesia vulpes, Babesia felis, Babesia cati, Babesia lengau, Babesia leo, Babesia microti, Babesia bovis, Babesia bigemina, Babesia caballi and a series of newly identified Babesia spp.[8][9][10][11][12][13][14][15].Notably, giant pandas, classified as carnivores within the Ursidae family [16], have exhibited cases of Babesia infection, especially among those that have been released into the wild in recent years.These cases have exhibited symptoms, including anaemia, jaundice and haemoglobinuria.Additionally, two cases of naturally occurring Babesia infection in wild giant pandas have been documented [7].Blood protozoa testing was subsequently conducted on all captive giant pandas at the giant panda base in Sichuan Province, as well as on wild giant pandas rescued from the wilderness.The results showed that 14 giant pandas tested positive for Babesia, with two wild giant pandas exhibiting typical clinical symptoms of babesiosis, and up to ten parasites were detected within a single erythrocyte, while the remaining positive giant pandas showed no clinical symptoms.
Considering the scarcity of previous research, this study explores the morphological features, phylogenetic relationships and taxonomic status of Babesia in giant pandas.This study aims to gain in-depth insights into the characterisation of this parasite within the Babesia genus and to offer guidance for future disease prevention and control efforts related to babesiosis.

Samples
This study investigated a total of 14 Babesia-positive giant pandas-eight from wild giant pandas across China (three from the Qionglai mountain range, three from the Xiaoxiangling mountain range, one from the Liangshan mountain range and one from the Minshan mountain range) and six from captive giant pandas in Sichuan Province (Fig. 1).

Morphological characterisation of the parasites
Blood smears were made using fresh whole blood collected from giant pandas and were subsequently stained with a Diff-Quick stain kit (Diff-Quick Stain Kit, Solarbio, Beijing, China).The stained blood smears were observed, and photographs were taken using a Zeiss Axio Imager M2 optical microscope (Carl Zeiss, Oberkochen, Germany) with an oil immersion magnification of 1000×; these images were processed using the Zeiss ZEN 2.3 lite software package (Carl Zeiss, Oberkochen, Germany).

Cloning and sequencing analysis of the mitogenome and 18S rRNA
Following the instructions provided in the DNeasy ® Blood and Tissue Kit manual (QIAGEN, Hilden, Germany), DNA extraction was carried out on 200 µl of EDTA-anticoagulated whole blood samples from giant pandas.A NanoDrop spectrophotometer (Nanodrop Technologies, Wilmington, DE) was used to measure the DNA concentrations after the extracted DNA samples were eluted using 200 µl of elution buffer.The isolated DNA was then preserved at -80 °C for use in subsequent molecular biology investigations.Both the 18S ribosomal RNA (18S rRNA) and the mitogenome were amplified using primers designed for the genus Babesia (Table 1).
The PCR amplicons were cloned into the pMD-19T vector (Takara, China) and transformed into Escherichia coli DH5α cells.Plasmid DNA was isolated and sent for sequencing by Sangon Biotech (Shanghai, China).Thereafter, the sequencing results were confirmed through sequence alignment using the Basic Local Alignment Search Tool (BLAST) on the National Center for Biotechnology Information (NCBI) website (https:// www.ncbi.nlm.nih.gov/ BLAST).

Phylogenetic analysis
Molecular Evolutionary Genetics Analysis (MEGA 5.0) was used to compare the amplified sequences of 18S rRNA, cytochrome b (cytb) and the complete mitogenome.The amplified sequences were aligned with those of other similar species listed in GenBank to construct a phylogenetic tree.During the sequence alignment stage, the ClustalW tool was used.The construction of the phylogenetic tree involved various analysis methods, including the maximum likelihood (ML) and neighbour-joining (NJ) methods.The ML tree was constructed using the general time reversible model (G + I) and calculated using the Akaike information criterion (AIC).The NJ tree was constructed with the Tamura-Nei model (G + I) according to the AIC [21,22].In the process of constructing the phylogenetic tree, 1000 bootstrap replicates were performed to assess the tree's reliability [23].DNAStar was utilised for pairwise genetic analysis of the 18S rRNA and cytb datasets, aimed at identifying possible evolutionary differences at the nucleotide level [22].The first evolutionary tree was constructed based on 61 (almost) fulllength sequences of the giant panda Babesia 18S rRNA and sequences of protozoa collected from other related species.Additionally, Toxoplasma gondii 18S rRNA   Vector: unknown.Haemaphysalis flava Neumann, 1897 is suspected [24,25].
Etymology: the species name was derived from the genus name of the type host.

Morphological description
In the stained blood smears, round to oval ring-shaped, paired pyriform, irregularly shaped and tetrad-shaped forms were observed (Fig. 2).There were varying numbers of parasites infecting erythrocytes, with individual ring-shaped parasites constituting the majority.Roundto-oval-shaped merozoites exist as single or double parasites within infected erythrocytes.The range of parasites infecting a single erythrocyte was between 1 and 10.The following were the size ranges of the different parasitic forms: round-to-oval ring-shaped
Furthermore, the cytb gene sequences of 11 positive giant panda blood samples were aligned with 25 protozoan cytb gene sequences obtained from the GenBank database to construct an evolutionary tree, with the H. canis cytb sequence serving as the outgroup.The evolutionary tree revealed three main clades; one clade represented the Babesia (s.s.) group, where all 11 cytb sequences of the giant panda Babesia formed a distinct clade and were situated within this group.They shared a close phylogenetic relationship with other Babesia cytb sequences, primarily including B. sp.pudui, B. sp.Dunhuang, B. sp.Coco, B. rossi, B. gibsoni, B. canis and some Babesia subspecies.Additionally, the other two main clades included Babesia (s.l.) and Theileriidae, which were relatively distantly related to the cytb clade of the giant panda Babesia and were situated closer to the outgroup (Fig. 4).
The mitogenome tree showed two primary clades: one representing the Babesia (s.s.) group, which was distinctively separate from the outgroup C. felis.Within Babesia (s.s.), the mitogenome sequence of the giant panda Babesia was closely related to that of B. gibsoni, B. rossi and B. canis.The remaining clades belonged to the Babesia (s.l.) group, positioned nearer to the outgroup C. felis, and primarily included B. conradae, B. microti, B. rodhaini and B. duncani (Fig. 5).

Discussion
To date, Babesia infections have been detected in animals from the Canidae, Felidae, Ursidae, and Procyonidae families worldwide, all of which are closely related to giant pandas.Among canids [31][32][33][34][35][36][37][38][39][40][41][42], including domestic dogs, wild dogs, foxes, raccoon dogs, black-backed jackals, grey wolves, coyotes and maned wolves, as well as felids such as domestic cats, wild cats, bobcats, caracals, ocelots, cheetahs, leopards, cougars, jaguars and lions, Babesia infections have been identified [43][44][45][46][47][48].Additionally, Babesia infections have been diagnosed in Ursidae animals, including black bears in Japan and the USA, brown bears in Japan, polar bears and sun bears [49][50][51][52][53][54][55].Moreover, reports of Babesia infection in Procyonidae animals have also been increasing steadily [56][57][58][59][60]. Recently, confirmed Fig. 3 Phylogenetic analysis of the nearly complete giant panda Babesia 18S rRNA sequences was conducted using the maximum likelihood method.The 18S rRNA sequence of T. gondii served as the outgroup.With a sequence length of 1679 bp, the general time reversible (G + I) model was used to construct the ML tree.The analysis did not include gaps or missing data.For each sequence, information on the host, the country of origin and the GenBank accession number are provided.Only bootstraps > 50% are shown, and the bootstrap values are based on 1000 repetitions (See figure on next page.)Fig. 3 (See legend on previous page.)cases of Babesia infection have been detected in wild giant pandas undergoing training [7].This study systematically classifies the Babesia species found in giant pandas, identifying it as a new taxonomic unit meeting the criteria for a new species according to the ICZN guidelines [26].According to ICZN standards, naming a newly discovered parasite involves several steps: providing a valid species description encompassing the morphological, biological and potential taxonomic characteristics; designating a type specimen; and assigning a Latinised name that conforms to international naming conventions to ensure uniqueness.This study meticulously described the shape, size, vector, type locality, type host, type material, ZooBank registration, designation and taxonomic status of Babesia in giant pandas.Consequently, the study named this species Babesia ailuropodae n. sp.The species name reflects the giant panda (Ailuropoda melanoleuca) as the primary natural host of this Apicomplexan parasite [7].
The morphological analysis in this study reveals that Babesia in giant pandas exhibits pleomorphism within erythrocytes, typically appearing as individual ringshaped parasites.This parasite predominantly parasitises the periphery of erythrocytes and shares parasitic characteristics similar to small Babesia species found in canids and felids [33,[61][62][63].Individual parasites of Babesia in giant pandas exhibit a size range of 1.365 µm × 1.574 µm in their various morphological forms.Additionally, B. ailuropodae n. sp. also appears in irregular forms,  B. ailuropodae n. sp.tends to be larger [46,67].The size of B. ailuropodae n. sp.overlaps significantly with that of B. gibsoni (1 µm × 3 μm), placing it in the category of medium-sized parasites [65,68].These distinct morphological characteristics indicate that the Babesia species found in giant pandas is unique.This finding presents an exciting prospect, as research on Babesia in Ursidae and Procyonidae has thus far been limited to epidemiological investigations, with detailed studies on morphology and systematic classification yet to be conducted.
Although a single-gene evolutionary tree may not fully capture the parasite's position in species evolution, combining multiple gene features with potentially diverse evolutionary histories can construct a more accurate phylogenetic tree that reflects the true relationships among related species [69].This study conducted a genetic analysis of the 18S rRNA and cytb genes in the giant panda Babesia to elucidate the evolutionary relationships of the newly identified species B. ailuropodae n. sp.The results indicated that Babesia isolates from giant pandas form a distinct evolutionary branch in different phylogenetic trees (Figs. 3 and 4), clearly distinguishing them from other Babesia species.This finding supports the idea that B. ailuropodae n. sp.could be unique to giant pandas because of its distinctive molecular evolutionary history.
In this study, four branches -Babesia (s.s.), Babesia (s.l.), Theileria and Hepatozoon -were visible in the phylogenetic tree built using the 18S rRNA gene sequence.Within Babesia (s.s.), B. ailuropodae n. sp.formed a sister clade along with the Babesia gathered from black bears and brown bears in Japan.It shares a high degree of similarity with the Babesia collected from domestic dogs in Japan and from the red panda in China [51,70].A geographical analysis revealed that the distributions of black bears, brown bears and domestic dogs in Japan do not overlap with those of giant pandas in China, which largely impedes inter-species disease transmission.However, this study revealed significant genetic similarities in the Babesia strains infecting these animals.The strong evolutionary kinship among giant pandas, black bears and brown bears, which are all members of the order Carnivora, likely explains this phenomenon.Despite their geographical separation, their genetic similarity suggests the potential for disease transmission among these species [71].H. flava, primarily found in East Asia, including China, Japan and Korea, is suspected to be a vector for giant pandas and has been detected in bear species in Japan, indicating its potential role in disease transmission [24,25].Therefore, we speculate that H. flava may serve as a common vector for Babesia infection.In this study, within the Babesia (s.s.) group, B. ailuropodae n. sp. and Babesia from the red panda were closely related, showing 99.23% sequence similarity in their 18S rRNA (OK524313), despite significant differences in their hosts' evolutionary lineages.Notably, the red panda, a member of the Ailuridae family within the superfamily Musteloidea, is evolutionarily distinct from the giant panda, which shares closer kinship with members of the bear family (Ursidae) [16,72,73].Despite their differences, giant pandas and red pandas have both inhabited the same geographical region and have undergone convergent evolution over time, thereby adapting similarly to environmental pressures and acquiring morphological and physiological similarities [74].Instances of Babesia infection in Procyonidae animals are well documented worldwide, and the similarity in genetic sequences of the Babesia derived from these animals corresponds to the phylogenetic relationships among their hosts [57-59, 75, 76].Simultaneously, the 18S rRNA sequences of Babesia in Japanese domestic dogs and those in giant pandas showed high similarity in this study, possibly due to ticks incidentally infecting Japanese domestic dogs, which may have originated from Japanese wild raccoons.This hypothesis was supported by the evolutionary tree analysis comparing Babesia sequences from Japanese domestic dogs with the 18S rRNA sequences from raccoons in Japan and the United States of America [14,59,70].These findings further support that Babesia infections in domestic dogs across various regions -Japan (AB118032, AF271082), China (HG328235, KP666168), Brazil (AY371196), India (L19079), Nigeria (JN982353), the USA (EU109717), and Europe (AY072926) -exhibit distinct evolutionary branches compared with B. ailuropodae n. sp.within Babesia (s.s.).Therefore, based on the 18S rRNA sequence classification analysis, the giant panda Babesia demonstrated a close phylogenetic relationship with Babesia species found in the bear (Ursidae), panda (Ailuridae), raccoon (Procyonidae) and dog (Canidae) families.
The cytb sequences of B. ailuropodae n. sp.differed by 1 to 53 base pairs from those of other Babesia species.Alignments of 82.6% with canine Babesia species and 77.92% with B. naoakii were obtained.This variability indicates its genetic independence in evolutionary development.Overall, the cytb gene sequences of 11 B. ailuropodae n. sp.formed a distinct branch, mirroring the branching pattern observed in the 18S rRNA analysis.This underscores the unique position of the giant panda Babesia within Babesia (s.s.).The cytb sequence of B. ailuropodae n. sp.showed close phylogenetic relationships with those of B. sp.Pudui (ON995403), B. sp.Dunhuang (MK962314), B. sp.Coco (KC207824), B. caballi (AB499086), B. gibsoni (OM933649) and B. rossi (KC207823).This genetic evolutionary analysis was consistent with the morphological characteristics of this species, supporting the classification of the giant panda Babesia as an independent taxonomic unit.Additionally, B. conradae (KC207826) and B. duncani (NC039721) within Babesia (s.l.) were found to be more distantly related to the outgroup, indicating a less close phylogenetic relationship with B. ailuropodae n. sp.This divergence may be attributed to environmental selection pressures between the parasites and their hosts, as well as inherent evolutionary differences among various parasitic species [62].
To date, the mitogenomes of most  [17,18,29,[77][78][79].The study of mitogenomes has contributed significantly to the provision of crucial information about the biological characteristics, inheritance and species categorisation of pathogens [27].In this study, the mitogenome of the giant panda Babesia was amplified, sequenced, assembled and then compared with those of other piroplasms from the GenBank database.The findings revealed that the mitogenome of the giant panda Babesia shares substantial similarity in size, structure and content with those of other Babesia species.It contains three protein-coding genes (cox1, cox3 and cytb) and six large subunit rRNA genes (LSU1-6), which are arranged linearly.However, attempts to amplify the two TIRs of the mitogenome using various methods, including highthroughput deep sequencing, inverted PCR, designing specific primers and optimising the PCR conditions, were unsuccessful in this study.This failure may be due to the extensive diversity and variability of TIRs among Babesia species within the phylum Apicomplexa, compounded by potential host-specific factors influencing the availability of TIRs in the mitogenomes of the studied Babesia species [17,30,80].The genetic analysis in this study revealed that the mitogenome of the giant panda Babesia is closely related to those of B. gibsoni, B. rossi, B. vogeli and B. canis, which form a sister branch.In contrast, it exhibits a more distant branching relationship with B. duncani, B. rodhaini, B. microti and B. conradae.

Conclusions
Based on morphological and molecular analyses (including mitogenome), we formally described a new species of piroplasmid infecting giant pandas in China, namely, Babesia ailuropodae n. sp.Further research is required to confirm the full host range and geographical distribution of the Babesia ailuropodae n. sp.vector.

Fig. 4
Fig.4 Phylogenetic analysis of the giant panda Babesia cytb gene sequences was conducted using the maximum likelihood method.The cytb sequence of H. canis served as the outgroup.With a sequence length of 1092 bp, the general time reversible (G + I) model was used to construct the ML tree.The analysis did not include gaps or missing data.For each sequence, information on the host, the country of origin and the GenBank accession number are provided.Only bootstraps > 50% are shown, and the bootstrap values are based on 1000 repetitions

Fig. 5
Fig. 5 Phylogenetic analysis of the giant panda Babesia mitogenome was conducted using the maximum likelihood method.The sequence of C. felis served as the outgroup.With a sequence length of 5609 bp, the TN93 + G model was used to construct the NJ tree.The analysis did not include gaps or missing data.Only bootstraps > 50% are shown, and the bootstrap values are based on 1000 repetitions

Table 1
Sequences of the PCR primers used to amplify the 18S rRNA and mitogenome from giant panda DNA samples For the analysis of the cytb gene, 36 sequences from related protozoan species were used, with the cytb gene sequences of Hepatozoon canis being used as outgroups.Moreover, in constructing the mitogenome tree, 17 related Babesia spp.sequences were used, with Cytauxzoon felis sequences serving as outgroups.

Table 2
Comparison of the identities between the complete 18S rRNA sequence (1679 bp) of the giant panda Babesia and other published Babesia sequences